The overall goal of these studies is to understand the endothelial nitric oxide synthase (eNOS) signaling system in health and disease. Regulation of endothelial NO production is a key determinant of blood pressure, platelet aggregation, and angiogenesis. The fundamental hypothesis of this research program is that eNOS post-translational modifications and protein-protein associations are essential determinants of NO-dependent signaling in the vascular wall. During the previous funding period, we investigated the relationships between eNOS subcellular targeting, acylation, and enzyme site-specific phosphorylation and dephosphorylation;identified novel kinase and phosphatase pathways involved in eNOS regulation;explored the inter-relationships of VEGF and sphingosine 1- phosphate (S1P) signaling pathways in eNOS regulation;developed and exploited siRNA-based methods to explore the role of caveolin in modulation of endothelial signal transduction;identified a key role for the small G protein Rac1 in control of kinase pathways that modulate eNOS;and discovered that eNOS undergoes reversible receptor-modulated S-nitrosylation. These discoveries set the stage for the proposed studies, which are organized into two Specific Aims. Aim 1 proposes studies to explore the hypothesis that nitrosylation of eNOS inhibits its enzyme activity. We will examine the relationships between cellular redox state and eNOS nitrosylation in cultured endothelial cells, and will determine the mechanisms whereby eNOS phosphorylation and subcellular targeting influence eNOS nitrosylation and denitrosylation. The proposed studies will generate and characterize transgenic mice that express the nitrosylation-deficient eNOS mutant to test the hypothesis that eNOS nitrosylation serves to mitigate excessive NO generation in the vessel wall. Studies proposed in Aim 2 will identify the mechanisms of Rac1 activation by cell surface receptors in endothelial cells, and will determine the molecular basis for Rac1 inhibition by caveolin. These studies will explore the effects of HMG CoA reductase inhibitors (statins) on caveolin-Rac1 signaling in the endothelium, and will identify the roles of Rac1 in NADPH oxidase vs. PI3-kinase activation in responses to VEGF and S1P. Our studies of the cellular and molecular mechanisms that regulate eNOS may lead to the identification of new points for pharmacological intervention.